The envelope glycoprotein (env) of human immunodeficiency virus (HIV-1) plays a central role in the pathogenicity of AIDS and the trimeric valency of the protein is important in determining the antigenic structure and immunogenicity of soluble analogues of env. Scanning transmission electron microscopy (STEM) has been applied to determine the subunit arrangement of unmodified and modified HIV-1 gp140. Macromolecular complexes were adsorbed onto thin carbon supports and plunge-frozen, freeze-dried and imaged in dark-field STEM at low electron dose. Images were analyzed quantitatively using a calibration standard of tobacco mosaic virus to determine the distributions of molecular weights and hence the oligomeric states of the proteins. Mass measurements revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 and especially the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Further measurements revealed that replacement of the N-terminal half of the gp41 segment of HIV-1 gp140 with the homologous motif of SIV was sufficient to confer efficient trimerization.